The shortage of donor organs for transplantation suggests a need to develop engineered tissue transplants. Proper in vitro vascularization, a key prerequisite for the development of functional engineered tissue constructs, would enable adequate mass exchange, gas supply, and functional mediator exchange in high- density tissue cultures. The impact of physical and mechanical factors supporting endothelial differentiation has been investigated, but not in three-dimensional (3D) co-culture models. We propose to address this gap in cellular models and technology model systems, by analyzing neo-vascularization in an organ-like environment in vitro designed to mimic human organogenesis and that can vary physical conditions, such as flow- and pressure changes in the rhythm of the heart rate. In the fetal liver in vivo, angiogenesis occurs in hematopoietic and hepatic tissues that develop together. In our cell model for enabling vascularization in vitro, we therefore propose to investigate second trimester human fetal liver derived endothelial progenitors within fetal parenchymal cells, which contribute to hematopoietic and hepatic tissue vascularization. In the culture technology model, we propose to apply physical forces to control vascular structure formation, shear stress, perfusion flow and pressure changes. Additionally we will investigate the effects of calcium liberating hydroxyapatite scaffolds that mimics natural bone on formation of hematopoietic vascular sinusoids in the stem cell niche. RFP transfection labeled progenitors (hemangioblasts and angioblasts) and non-endothelial fetal liver cells will be cultured in 3D perfusion and the response to various physical-mechanical cues determined. Harvested cells will be analyzed by histology, flow cytometry, and gene expression, and compared to prenatal organ explants and postnatal organ tissues in vivo. The prior labeling of hemangioblasts will allow us to selectively distinguish between original hemangioblasts, endothelial- and non-endothelial cell types. The bioreactor model provides four independent interwoven hollow fiber compartments, enabling 3D perfusion with low gradients by decentral mass exchange and integral oxygenation. This has been proven to support vascularized tissue-like structure formation at high cell densities. We have already demonstrated that our 3D perfusion bioreactors support the spontaneous neo-tissue formation with neo-vascular hepatic structures and functionality in the laboratory and in clinical application for extracorporeal liver support. The innovation of our project is the specific experimental model that mimics the mass exchange in the native organ environment, allowing the fate of labeled fetal vascular progenitors to be studied during tissue formation, depending on different physical conditions. The project outcome will contribute to our understanding of the role of bioengineered supports and physical forces in establishing functional 3D engineered neo-vascular constructs in hematopoietic and hepatic tissues. (End of Abstract)